This invention relates in general to gel chromatography and relates more particularly to methods of producing a gel-packed column for use in electrophoresis chromatography. A gel is a cross-linked polymer network swollen in a solvent medium.
The field of chromatography is discussed in A. Braithwaite and F. J. Smith, Chromatographic Methods, Chapman and Hall, Fourth Edition. A discussion of electrophoresis is presented in U.S. Pat. No. 4,675,300 entitled Laser-Excitation Fluorescence Detection Electrokinetic Separation issued to Richard N. Zare on Jun. 23, 1987. In chromatography, two components in a sample are separated by passing the sample through a medium in which one component travels at a faster rate than the other component. In general, this is achieved by utilizing a mobile phase and a stationary phase. The mobile phase can be a gas or a liquid and the stationary phase can be a solid or can be a liquid supported on a solid or on a gel. It is very common today to include one or more sensors along the chromatography column to detect the samples as they pass such sensors. For example, a spectrophotometer can be connected to the chromatograph to not only detect the passage of sample peaks, but also to measure the spectral absorption of each peak to identify the sample component or components in each peak.
In ion exchange chromatography, the samples have affinities for both the mobile phase and the stationary phase. A sample having a relatively greater affinity for the mobile phase than for the stationary phase will spend a greater fraction of its time in the chromatograph attached to the mobile phase and therefore will travel along the chromatograph at a greater speed than does another sample component having a smaller relative affinity for the mobile phase. Ion exchange chromatography therefore separates samples according to their relative affinity for these two phases. Various choices of mobile and stationary phases can be made to optimize the separation of the expected sample components.
Gel chromatography has been found to be particularly useful for separating biological molecules from organic mixtures (see, Braithwaite and Smith, sections 4.5, 4.8 and 4.9). Gels are produced by a polymerization process that produces porous structures, each consisting of a cross-linked polymer having a pore size that is regulated by controlling the amount of cross-linking. Gels typically contain on the order of 0.5-15 weight % cross-linked polymer and 55-99.5 weight % solvent. Such a three-dimensional lattice allows diffusion of molecules through the lattice at rates that are dependent on the relative sizes of the molecules to the pores in the lattice. Such separation by size and shape is important in biological chemistry because biological molecules exhibit a greater range of size and shape than of chemical affinity as is important in ion-exchange chromatography.
The most common geometries for the gels are gel slabs and gel columns. The passage of electric current in traditional systems with gel slabs and columns having diameters and thicknesses on the order of millimeters and greater, produces an undesired amount of Joule heating. Such heating can distort the gel structure, thereby interfering with the sample component separation process. To avoid the need for cooling systems to remove such heat, the trend has been away from traditional chromatography columns and toward the use of capillaries having diameters on the order of tens to hundreds of microns. The increased surface-to-volume ratio enhances heat removal and the smaller bore decreases the amount of sample required for a measurement, improves the measurement accuracy and increases the speed of measurement.
In gel ion-exchange chromatography and gel permeation chromatography, the gel is generally formed into beads that are on the order of 10's to 100's of microns in diameter with a pore size that is on the order of the size of the sample particles that are to be separated (typically on the order of a few to millions of Angstroms). These beads are poured into one end of the chromatograph column as a slurry of solvated beads and are prevented from exiting the other end of the column by a frit (i.e., a porous matt) attached to the other end of the column to restrain the beads. The bead size is selected to be as small as possible while still allowing an adequate flow rate of sample and eluent through the chromatograph. The amount of compaction of the mass of beads is kept low enough that the larger molecules have a free pathway to travel around the beads.
In gel permeation chromatography, only the sieve-like structure of the gel is utilized to separate the sample components so that separation is substantially independent of the chemical affinities of the sample components. In gel ion-exchange chromatography, both the sieve-like nature of a gel and the chemical affinity of electrolytes in the chromatograph are utilized to produce the sample separations.
In gel ion-exchange chromatography, the sample is carried along the chromatograph by an electrolyte having ions that have a chemical affinity for the gel. Sample ions on the gel are displaced by ions in the mobile phase of the same sign of charge as the displaced ions. The ion-exchange mechanism takes place in the thin film of solvent at the surface of the bead, including those portions of the surface adjacent to the pores of the bead. Ions comparable to or larger than the pore size are excluded from the interior of the resin so that only ions smaller than the pore size diffuse within the lattice flamework of a bead. Such small ions therefore experience the large surface area interior of a bead and therefore experience a large number of ion exchange interactions with the resin or gel, thereby travelling along the chromatograph at a slower rate than the ions that are larger than the pore size. These smaller atoms experience the full ion-exchange capacity of the resin or gel, which is defined to be the amount of charged groups per gram of dry resin or gel. Any compounds that are completely excluded from the gel will not be separated from each other and any compounds that completely penetrate the gel will not be separated from each other.
The ion exchange process is an equilibrium process in which the affinity between the exchange ion and the bead surface is a function of the chemistry of both the exchange ion and the bead. Ions with a large affinity for the bead travel along the chromatograph more slowly than ions with smaller affinity so that the rate of travel is dependent on such affinity as well as on the ratio between ion and pore sizes. Depending on the pH, the sample ions can attach to the beads strongly enough that they form a substantially stationary band within the chromatograph. An eluent with a pH sufficient to displace the bound sample ions is then used to wash the sample along the chromatograph. The bound ions will be eluted in descending order of their affinity for the beads. The available ion-exchange capacity is the actual capacity that results under experimental conditions and is dependent on the accessibility of functional groups, on eluent concentration, on ionic strength and pH, on the nature of the counter ions and on the strength of the ion exchanger and its degree of cross-linkage.
In FIG. 1 is illustrated an apparatus for electrophoretic separation chromatography. A first buffer solution 11 is contained in a container such as beaker 13 and a second buffer solution 12 is contained in a second container such as beaker 14. Each end of a capillary 15 is immersed in one of these two beakers and a voltage source 16 produces a voltage difference between these solutions on the order of 5-30 kV and a current through capillary 15 on the order of 1-25 .mu.A. Capillary 15 has an inside diameter on the order of 2-500 .mu.m and a length that is typically in the range from 20 cm to a meter. Although the typical range of capillary diameters is 2-500 .mu.m, other diameters can also be used. In particular, the method described below is also useful for larger diameter capillaries. However, for such larger diameter capillaries, other methods of filling the capillaries are available.
In FIG. 2 is illustrated in greater detail a small section of capillary 15. The interior cavity 20 of capillary 15 is filled with a conductive liquid referred to as the "support electrolyte". Wall 21 of capillary 15 adsorbs ions 22 (which in this embodiment are negative, but for other choices of support electrolyte and wall 21 can be positive), thereby leaving an excess of positively charged ions 23 in the body 24 of the support electrolyte. Voltage source 16 produces an electric field E that drives positively charged fluid body 24 toward the cathode of voltage source 16. In addition, positively charged particles are driven toward the cathode and negatively charged particles, such as particle 25, are driven toward the anode of voltage source 16. Sample is loaded into capillary 15 by immersing the inlet end of the capillary into a vial containing the sample and briefly turning on the electric field to draw some of the sample into the capillary. The inlet end of the capillary is then reinserted into beaker 13 and the electric field is turned on to draw sample ions from beaker 13 through capillary 15.
Many biological molecules are amphoteric so that the pH of the support electrolyte can be selected to control the sign of charge on selected sample components. Because of this ability to control the charge of sample components, some sample component separation can be achieved by this control of the charge of the sample components. However, because biological molecules have a greater variation in size and shape than in charge, it is advantageous to fill interior cavity 20 of capillary 15 with a gel having a pore size selected to separate selected components of the sample as the primary separation mode. Unfortunately, for a variety of reasons, it is difficult to achieve a continuous gel within cavity 20.
In gel electrophoresis chromatography, the gel is typically produced inside the capillary by mixing the gel precursors (typically including reactive monomers or prepolymers, one or more crosslinking agents, polymerization catalyst, polymerization initiator and other additives that may be useful during the separation process such as surfactants and denaturizers), filling the capillary with this mixture and allowing the gel to cure within the capillary. Unfortunately, in addition to the gel, this process leaves in the gel residues that can interfere with the chromatographic separation of sample components and lead to premature breakdown of the gel. Because of the extreme length to diameter ratio of capillary 15, these residues are not easily removed from the gel by flow of eluent through the capillary. Indeed, in electrophoresis, there is almost no flow of eluent through the gel. The only species that exhibit significant motion are the ionic species.
Another problem is that the gel generally shrinks by a few percent volume when it cures so that the gel tends to pull away from the walls of the capillary. As a result of this, when the electric field is turned on to push sample ions through the capillary, the gel tends to be pushed along and out of the capillary due to ionic groups associated with the gel. To prevent this, it is common to treat the inside surface of the capillary wall and/or to add to the gel precursor a coupling agent, such as silane, to bond the gel to the capillary wall.
An additional problem is that voids sometimes occur in the gel. Such voids are more readily produced in gels that are bonded to the capillary or column wall because they are prevented from pulling away from the wall as they shrink during curing. These voids present obstacles to the ionic flow and can introduce inhomogeneities in the process that degrade resolution. If such a void extends entirely across the internal diameter of the capillary, there will be a complete break in the current path and electrophoresis will be stopped.
To overcome the problem of voids, in one gel formation process, the capillary is first filled with the gel precursor. Preferably, the gel precursor is at a reduced temperature that inhibits the chemical reaction that results in formation of the gel. The capillary is then either heated or exposed to radiation in a narrow zone to cure the gel precursor within that zone. This zone is then moved along the capillary to cure the gel along the entire length of the capillary. By use of this moving zone of curing, the still mobile gel precursor can flow toward the cured zone to compensate for the shrinkage that occurs during curing. Unfortunately, this moving zone process is a slow process that is difficult to control and that significantly increases the time required to produce a gel within the capillary.